•Gene electrotransfer can be achieved with high frequency bipolar microsecond pulses.•Gene electrotransfer efficiency depends on plasmid concentration.•Number of transferred plasmid copies is higher with longer monopolar pulses.•No correlation between size of plasmid aggregates and transfection efficiency. High-frequency bipolar pulses (HF-BP) have been demonstrated to be efficient for membrane permeabilization and irreversible electroporation. Since membrane permeabilization has been achieved using HF-BP pulses we hypothesized that with these pulses we can also achieve successful gene electrotransfer (GET). Three variations of bursts of 2 µs bipolar pulses with 2 µs interphase delay were applied in HF-BP protocols. We compared transfection efficiency of monopolar micro and millisecond pulses and HF-BP protocols at various plasmid DNA (pDNA) concentrations on CHO – K1 cells. GET efficiency increased with increasing pDNA concentration. Overall GET obtained by HF-BP pulse protocols was comparable to overall GET obtained by longer monopolar pulse protocols. Our results, however, suggest that although we were able to achieve similar percent of transfected cells, the number of pDNA copies that were successfully transferred into cells seemed to be higher when longer monopolar pulses were used. Interestingly, we did not observe any direct correlation between fluorescence intensity of pDNA aggregates formed on cell membrane and transfection efficiency. The results of our study confirmed that we can achieve successful GET with bipolar microsecond i. e. HF-BP pulses, although at the expense of higher pDNA concentrations.